Genomic full-length sequence of the HLA-B*13:64 allele was identified in a Chinese bone marrow donor.

Genomic full-length sequence of HLA-B*13:64 was identified in a Chinese individual by sequence-based typing.

A novel HLA-A*33 allele, HLA-A*33:131, identified by sequencing-based typing.

The novel allele HLA-A*33:131 was identified in a Chinese individual using the sequencing-based typing method.

A novel HLA class I allele, HLA-B*13:02:21, identified by sequence-based typing in a Chinese individual.

HLA-B*13:02:21 has one nucleotide substitution, C > T(GAC to GAT) compared with B*13:02:01.

[Sequence Analysis of A Novel HLA-B*13:68 Allele].

OBJECTIVE: The purpose of this research was to identify a novel HLA-B allele in Chinese population. METHODS: The HLA typing of bone marrow donors was performed by PCR-SBT. The ambiguous novel HLA allele was confirmed with GSSP method. RESULTS: The sequence of a sample was different from all alleles in the HLA-B databases. The sequence analysis showed that it differed from the closet matching allele HLA-B*13:01:01 at one nucleotide substitution, 137 T > C in Exon2, which resulted in an amino acid change from Phenylalanine (Phe) to Serine (Ser) at codon 22. CONCLUSION: A novel allele was identified and named as HLA-B*13:68 by the WHO Nomenclature Committee.

[Preparation and identification of human soluble sPD-L1 and its antibodies].

This study reports the preparation and identification of soluble programmed death-1 (PD-1) ligand-1 (sPD-L1) and its antibodies of mouse origin. Immobilized metal ion affinity chromatography was used to perform on-column refolding with simultaneous purification of denatured sPD-L1, and soluble sPD-L1 with purity of 95% was obtained. The purified sPD-L1 was verified by immunoblotting using a commercial goat-anti-human PD-L1 antibody. An ELISA-based assay showed that it also had high binding activity for its cognate receptor PD-1. Furthermore, mouse anti-sPD-L1 antiserum of high titer was raised using the purified sPD-L1 as an immunogen, and the specific IgG antibodies were purified using sPD-L1-HiTrap affinity chromatography. In addition, a sensitive sandwich ELISA was established using the purified IgG antibodies together with the commercial goat antibodies. In conclusion, the preparation of soluble sPD-Ll and its antibodies provide the basis for detection of the potential anti-PD-L1 antibodies and soluble PD-L1 in humans as well as for further investigation of its in vivo bioactivities and characterization of its potential receptors.

High frequencies cytomegalovirus pp65(495-503)-specific CD8+ T cells in healthy young and elderly Chinese donors: characterization of their phenotypes and TCR Vbeta usage.

Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus which persists lifelong after primary infection and can lead to a significant disease in the immunocompromised individuals. CD8(+) T cells are believed to play a crucial role in both the elimination of active infection and maintenance of HCMV latency. Large expansions of CD8(+) T cells specific for a single epitope of HCMV have been well documented in Caucasoid population. To date, no similar study has been performed in Chinese populations. Here we report the characteristics of HCMV-specific CD8(+) T cells in healthy young and elderly Chinese donors using pp65(495-503)-loaded HLA-A*0201 tetramers. Cells were stained with a combination of the tetramers and antibodies for CD28 and CD57 or a panel of TCR Vbeta and analyzed by three-color flow cytometry. The frequencies of pp65(495-503)-specific T cells within total CD8(+) T cell population were between 0.14 and 6.84% (mean 2.45%) in the young donors and were from 0.33 to 6.89% (mean 1.95%) in the elderly donors, respectively. There was no significant difference between the two groups. The expression of CD28 was decreased whereas CD57 expression was increased in tetramer-negative CD8(+) T cells in the elderly when compared with the young group. However, neither of these changes was found within tetramer-positive cell populations. Moreover, TCR Vbeta usage within tetramer-positive population was predominated by certain TCR Vbeta subsets. These results demonstrate that large expansions of HCMV-specific CD8(+) T cells with certain subsets TCR Vbeta exist both in the healthy young and in the elderly Chinese individuals, which may play a role in the maintenance of virus latency but have potential detrimental influence on the immune responses to other pathogens or vaccinations.

[Quantification and phenotypic analysis of hCMV specific CTL in peripheral blood from HLA-A2+ donors].

AIM: To optimize tetramer staining condition using HLA-A*0201 tetramer (A2-NLV tetramer) loaded with NLV peptide (pp65(495-503)) derived from structural protein pp65 of human cytomegalovirus and to investigate its application in phenotyping of specific cytotoxic T lymphocytes (CTL). METHODS: Peripheral blood from HLA-A2(+) donors was first stained with A2-NLV tetramer/PE under different conditions and then labeled with anti-CD3-FITC and anti-CD8-APC. The stained samples were analyzed with flow cytometry to find out the optimized staining condition. Meanwhile, the phenotype and activation antigen expression were determined. RESULTS: Tetramer staining with whole blood was superior to peripheral blood mononuclear cells. The optimized condition for tetramer staining was incubating 100 muL of whole blood with 0.3 mug of A2-NLV tetramer for 1 h at 4 degrees Celsius. Under this condition the specific staining was strong while unspecific staining of CD8(-) T cells was quite weak. Phenotypic analysis under this condition showed that the ratio of CD28 positive A2-NLV tetramer specific CTL was lower than that of nonspecific CTL, whereas the ratio of CD57 positive specific CTL was higher than that of nonspecific CTL. CD25 molecules were only expressed on the activated specific CTL. CONCLUSION: The optimized tetramer staining condition can increase the specificity of tetramer staining and decrease unspecific binding, therefore it is applicable for phenotyping and functional analysis of antigen-specific CTL.